A novel metalloenzyme with two distinct isoforms has been identified in the methionine salvage pathway of Klebsiella pneumoniae. The enzyme, presently named E2, has been cloned and expressed in E. coli. Surprisingly, induction of the cloned gene results in overexpression of two distinct isoforms, E2 and ET, which are separable by chromatography. The two forms differ (at least in composition) only by their metal ion contents. Both enzymes catalyze the oxidative degradation of 1,2-dihydroxy-3-keto-5-methylthiopentene anion, but the product of the degradation depends upon the identity of the metal ion bound in the active site. Ni 12 bound in the active site results in the formation of carbon monoxide and formate from the degradative process. If Fe'2 is bound to the enzyme, only formate is produced. We have prepared a sample of the fully "C,"N-enriched Ni,2_Containing enzyme and have acquired spectra using the standard repertoire of triple resonance experiments (HN(CO)CA, HNCA, HNCACB, CBCA(CO)NH) and 13C-edited experiments (HCCH-TOCSY, 13C-filtered NOESY). Nearly complete 'H, "C and "N resonance assignments have been made for the native Ni" containing E2 enzyme, and a global fold for the enzyme has been determined. There is preliminary evidence that structural differences between the two isoforms may be due to the presence (in E2) or absence (in E2') of a disulfide bridge between the only two cysteines in the protein.